Lung cancer is the leading cause of cancer death among US Veterans. The most effective therapy is surgical resection, but lung cancer recurs in approximately 50% of patients, most commonly as metastatic disease. This suggests that micrometastatic disease is often already present at the time of surgery, but below the level of detection of our current imaging studies. This is consistent with reports of circulating tumor cells in patients with stage I non-small cell lung cancer (NSCLC). Although metastatic behavior is often considered a late event, these clinical findings suggest that the metastatic process is also operative early in the pathogenesis of the disease. These clinical observations are consistent with recent laboratory-based investigations indicating that dissemination may occur during early tumor development. Furthermore, recent studies implicate the genetic program associated with epithelial-mesenchymal transition (EMT), not only in driving metastatic behavior, but also in transforma-tion and stem cell expansion. Our preliminary studies indicate that key genes in the EMT program are responsible for both transformation and enhanced motility of human bronchial epithelial cells (HBECs). In addition, we find that HBECs expressing genes associated with EMT or common NSCLC driver mutations show marked heterogeneity in their capacity for cell motility. We hypothesize that enhanced epithelial cell motility is operative during premalignancy and is a driver of early metastatic dissemination. The proposed research will elucidate the fundamental mechanisms involved in epithelial cell motility and their potential impact on disease onset and progression by coupling novel in vitro and in vivo models of human lung carcinogenesis and an innovative motility-based cell isolation technique. We anticipate that the proposed investigations will yield a more complete understanding of the molecular determinants of lung cancer pathogenesis, which will stimulate development of more effective chemopre-vention and early detection. Here, we have focused attention on the transcriptional repressor prototype SNAI1 (Snail) as a molecular driver of the high velocity phenotype, because we find that it: 1) is up-regulated in human NSCLC and pulmonary premalignancy in situ; 2) portends a poor prognosis when elevated in early stage NSCLC and mediates tumor-promoting phenotypes in lung cancer, including angiogenesis; 3) is a driver of the stem cell expansion, transformation, and metastatic behavior of HBECs in vitro and in vivo; and 4) is a mediator of the enhanced motility and deformability of HBECs in vitro. As these properties are important for metastatic behavior, we hypothesize that motility may also facilitate lung carcinogenesis and early dissemination via both Snail/EMT-dependent and -independent expression of motility-related genes. The studies proposed in Aims 1 and 2 will identify the motility-based pathway intermediaries that play a role in early metastatic behavior and carcinogenesis in in vitro human models. By determining the prevalence of the leading motility gene candidates in human premalignant lung lesions, subAim 2B will also ensure that our subsequent detailed mechanistic studies are focused on novel drivers of motility that are relevant to the clinical situation they ae intended to investigate (pulmonary premalignancy). Finally, Aim 3 seeks to determine the contribution of the clinically-relevant motility-candidates identified in Aims 1 and 2 to growth an early metastatic seeding in vivo. By coupling 1) innovative approaches to single-cell motility and deformability analysis with 2) a novel model of premalignancy (that includes the capacity to induce transformation to cancer in a human system in mice) and 3) a rare bank of pulmonary premalignant lesions (that ensures the clinical relevance of all findings), we are uniquely poised to identify the mechanisms underlying motility that are critical for early metastatic behavior in situ. This research is a prerequisite for future studies that focus on new biomarkers of disease onset and new targets for early detection, disease monitoring, and chemoprevention for lung cancer.